L-Lactic acid production by combined utilization of agricultural bioresources as renewable and economical substrates through batch and repeated-batch fermentation of Enterococcus faecalis RKY1.

Enterococcus faecalis RKY1 was used to produce l-lactic acid from hydrol, soybean curd residues (SCR), and malt. Hydrol was efficiently metabolized to l-lactic acid with optical purity of >97.5%, though hydrol contained mixed sugars such as glucose, maltose, maltotriose, and maltodextrin. Combined utilization of hydrol, SCR, and malt was enough to sustain lactic acid fermentation by E. faecalis RKY1. In order to reduce the amount of nitrogen sources and product inhibition, cell-recycle repeated-batch fermentation was employed, where a high cell mass (26.3g/L) was obtained. Lactic acid productivity was improved by removal of lactic acid from fermentation broth by membrane filtration and by linearly increased cell density. When the total of 10 repeated-batch fermentations were carried out using 100g/L hydrol, 150g/L SCR hydrolyzate, and 20g/L malt hydrolyzate as the main nutrients, lactic acid productivity was increased significantly from 3.20g/L/h to 6.37g/L/h.

Cell-recycle continuous fermentation of Enterococcus faecalis RKY1 for economical production of lactic acid by reduction of yeast extract supplementation.

Both lactic acid productivity and cell growth were linearly correlated with yeast extract supplementation in batch fermentation. During conventional continuous operation, although fresh feed was introduced into the bioreactor with a significantly low dilution rate (0.04 h(-1)), the amount of yeast extract employed was not enough to maintain the growth of microorganism. However, when the fresh feed contained 100 g/l glucose and 2 g/l yeast extract during cell-recycle continuous operation at a dilution rate of 0.04 h(-1), more than 90 g/l lactic acid was continuously produced, with the average productivity of 3.72 g/l·h. In this experiment, 82 g of yeast extract (77% of reduction yield) could be reduced for the production of 1 kg of lactic acid compared with batch fermentation of a similar volumetric productivity.

Immobilization of cellulase from newly isolated strain Bacillus subtilis TD6 using calcium alginate as a support material.

Bacillus subtilis TD6 was isolated from Takifugu rubripes, also known as puffer fish. Cellulase from this strain was partially purified by ammonium sulphate precipitation up to 80% saturation, entrapped in calcium alginate beads, and finally characterized using CMC as the substrate. For optimization, various parameters were observed, including pH maximum, temperature maximum, sodium alginate, and calcium chloride concentration. pH maximum of the enzyme showed no changes before and after immobilization and remained stable at 6.0. The temperature maximum showed a slight increase to 60 °C. Two percent sodium alginate and a 0.15 M calcium chloride solution were the optimum conditions for acquisition of enzyme with greater stability. K (m) and V (max) values for the immobilized enzyme were slightly increased, compared with those of free enzyme, 2.9 mg/ml and 32.1 µmol/min/mL, respectively. As the purpose of immobilization, reusability and storage stability of the enzyme were also observed. Immobilized enzyme retained its activity for a longer period of time and can be reused up to four times. The storage stability of entrapped cellulase at 4 °C was found to be up to 12 days, while at 30 °C, the enzyme lost its activity within 3 days.

The statistically optimized production of poly(gamma-glutamic acid) by batch fermentation of a newly isolated Bacillus subtilis RKY3.

For the production of poly(gamma-glutamic acid), a newly isolated Bacillus sp. RKY3 was phylogenetically identified as Bacillus subtilis based on its 16S rRNA gene sequence. The culture medium for the production of poly(gamma-glutamic acid) by B. subtilis RKY3 was optimized statistically. The parameters significantly affecting poly(gamma-glutamic acid) production were found to be glycerol, glutamic acid, yeast extract, and K(2)HPO(4). A further advanced statistical approach, central composite design, found the optimum levels of the screened variables as follows (gl(-1)): glycerol 17.6, glutamic acid 59.6; yeast extract 2.7; K(2)HPO(4) 2.3. The predicted response as poly(gamma-glutamic acid) production under the statistically optimized conditions was 48.5 g l(-1), which was only 0.4% different from the maximum poly(gamma-glutamic acid) concentration (48.7 g l(-1)) observed at the validation experiment using 7-l lab-scale fermentor containing 3 l of working volume.

Lactic acid production by Lactobacillus sp. RKY2 in a cell-recycle continuous fermentation using lignocellulosic hydrolyzates as inexpensive raw materials.

Continuous lactic acid fermentations were conducted using lignocellulosic hydrolyzates and corn steep liquor as inexpensive raw materials. Lactic acid concentrations decreased with increases in the dilution rate, whereas the residual substrate concentrations increased. However, lactic acid yields were maintained at more than 0.90 g g(-1) over all cases experimented. The cell-recycle cultivation system exerted positive effects on fermentation efficiency, including volumetric productivity, which is attributable to the retention of cells in the bioreactor. The cell-recycle continuous fermentation of lignocellulosic hydrolyzates yielded a lactic acid productivity of 6.7 g l(-1) h(-1) for a dilution rate of 0.16 h(-1) using 30 g l(-1) of corn steep liquor and 1.5 g l(-1) of yeast extract as nutrients. The productivity (6.7 g l(-1) h(-1)) acquired by the cell-recycle continuous fermentation of lignocellulosic hydrolyzates was 1.6 times higher than the lactic acid productivity yielded in the continuous fermentation without cell-recycle system.

Optimization of alkaline protease production by batch culture of Bacillus sp. RKY3 through Plackett-Burman and response surface methodological approaches.

The proteolytic enzymes are the most important group of commercially produced enzymes. The production of alkaline protease was optimized using a newly isolated Bacillus sp. RKY3. The fermentation variables were selected in accordance with the Plackett-Burman design and were further optimized via response surface methodological approach. Four significant variables (corn starch, yeast extract, corn steep liquor, and inoculum size) were selected for the optimization studies. The statistical model was constructed via central composite design (CCD) using three screened variables (corn starch, corn steep liquor, and inoculum size). An overall 2.3-fold increase in protease production was achieved in the optimized medium as compared with the unoptimized basal medium. Enzyme activity increased significantly with optimized medium (939 u ml(-1)) when compared with unoptimized medium (417 u ml(-1)).

Bacterial cellulose production by Gluconacetobacter sp. PKY5 in a rotary biofilm contactor.

A rotary biofilm contactor (RBC) inoculated with Gluconacetobacter sp. RKY5 was used as a bioreactor for improved bacterial cellulose production. The optimal number of disk for bacterial cellulose production was found to be eight, at which bacterial cellulose and cell concentrations were 5.52 and 4.98 g/L. When the aeration rate was maintained at 1.25 vvm, bacterial cellulose and cell concentrations were maximized (5.67 and 5.25 g/L, respectively). The optimal rotation speed of impeller in RBC was 15 rpm. When the culture pH in RBC was not controlled during fermentation, the maximal amount of bacterial cellulose (5.53 g/L) and cells (4.91 g/L) was obtained. Under the optimized culture conditions, bacterial cellulose and cell concentrations in RBC reached to 6.17 and 5.58 g/L, respectively.

Synthesis of poly(sorbitan methacrylate) hydrogel by free-radical polymerization.

Hydrogels are materials with the ability to swell in water through the retention of significant fractions of water within their structures. Owing to their relatively high degree of biocompatibility, hydrogels have been utilized in a host of biomedical applications. In an attempt to determine the optimum conditions for hydrogel synthesis by the free-radical polymerization of sorbitan methacrylate (SMA), the hydrogel used in this study was well polymerized under the following conditions: 50% (w/v) SMA as monomer, 1% (w/w) alpha, alpha'-azo-bis(isobutyro-nitrile) as thermal initiator, and 1% (w/w) ethylene glycol dimethacrylate as cross-liking agent. Under these conditions, the moisture content of the polymerized SMA hydrogel was higher than in the other conditions. Moreover, the moisture content of the poly(SMA) hydrogel was also found to be higher than that of the poly(methyl methacrylate [MMA]) hydrogel. When the Fourier transform-infrared spectrum of poly(SMA) hydrogel was compared with that of poly(MMA) hydrogel, we noted a band at 1735-1730/cm, which did not appear in the Fourier transform-infrared spectrum of poly(MMA). The surface of the poly(SMA) hydrogel was visualized through scanning electron microscopy, and was uniform and clear in appearance.

Production of lactic acid from cheese whey by batch and repeated batch cultures of Lactobacillus sp. RKY2.

The fermentative production of lactic acid from cheese whey and corn steep liquor (CSL) as cheap raw materials was investigated by using Lactobacillus sp. RKY2 in order to develop a cost-effective fermentation medium. Lactic acid yields based on consumed lactose were obtained at more than 0.98 g/g from the medium containing whey lactose. Lactic acid productivities and yields obtained from whey lactose medium were slightly higher than those obtained from pure lactose medium. The lactic acid productivity gradually decreased with increase in substrate concentration owing to substrate and product inhibitions. The fermentation efficiencies were improved by the addition of more CSL to the medium. Moreover, through the cell-recycle repeated batch fermentation, lactic acid productivity was maximized to 6.34 g/L/h, which was 6.2 times higher than that of the batch fermentation.

Production of bacterial cellulose by Gluconacetobacter sp. RKY5 isolated from persimmon vinegar.

The optimum fermentation medium for the production of bacterial cellulose (BC) by a newly isolated Gluconacetobacter sp. RKY5 was investigated. The optimized medium composition for cellulose production was determined to be 15 g/L glycerol, 8 g/L yeast extract, 3 g/L K2HPO4, and 3 g/L acetic acid. Under these optimized culture medium, Gluconacetobacter sp. RKY5 produced 5.63 g/L of BC after 144 h of shaken culture, although 4.59 g/L of BC was produced after 144 h of static culture. The amount of BC produced by Gluconacetobacter sp. RKY5 was more than 2 times in the optimized medium found in this study than in a standard Hestrin and Shramm medium, which was generally used for the cultivation of BC-producing organisms.

Production of lactic acid from cheese whey by batch and repeated batch cultures of Lactobacillus sp. RKY2.

The fermentative production of lactic acid from cheese whey and corn steep liquor (CSL) as cheap raw materials was investigated by using Lactobacillus sp. RKY2 in order to develop a cost-effective fermentation medium. Lactic acid yields based on consumed lactose were obtained at more than 0.98 g/g from the medium containing whey lactose. Lactic acid productivities and yields obtained from whey lactose medium were slightly higher than those obtained from pure lactose medium. The lactic acid productivity gradually decreased with increase in substrate concentration owing to substrate and product inhibitions. The fermentation efficiencies were improved by the addition of more CSL to the medium. Moreover, through the cell-recycle repeated batch fermentation, lactic acid productivity was maximized to 6.34 g/L/h, which was 6.2 times higher than that of the batch fermentation.

Production of bacterial cellulose by Gluconacetobacter sp. RKY5 isolated from persimmon vinegar.

The optimum fermentation medium for the production of bacterial cellulose (BC) by a newly isolated Gluconacetobacter sp. RKY5 was investigated. The optimized medium composition for cellulose production was determined to be 15 g/L glycerol, 8 g/L yeast extract, 3 g/L K2HPO4, and 3 g/L acetic acid. Under these optimized culture medium, Gluconacetobacter sp. RKY5 produced 5.63 g/L of BC after 144 h of shaken culture, although 4.59 g/L of BC was produced after 144 h of static culture. The amount of BC produced by Gluconacetobacter sp. RKY5 was more than 2 times in the optimized medium found in this study than in a standard Hestrin and Shramm medium, which was generally used for the cultivation of BC-producing organisms.

Recovery of lactic acid by repeated batch electrodialysis and lactic acid production using electrodialysis wastewater.

Repeated batch electrodialysis for lactic acid recovery was investigated using lactic acid solution and fermentation broth. In both cases, lactate fluxes averaged more than 7.0 moles/m2.h, lactate recovery reached more than 99% for all the batch runs, and specific energy consumption per unit lactate transported was lower than 0.25 kWh/kg-lactate. When electrodialysis wastewater was used as a fermentation medium, supplemented with 100 g/l glucose, up to 92.4 g/l lactic acid was produced with a productivity of 0.67 g/l.h. In addition, when electrodialysis wastewater was supplemented with 150 g/l whole-corn flour hydrolyzate and 5 g/l corn steep liquor, 2.5-fold and 1.8-fold increases in lactic acid productivity and maximum cell growth, respectively, were achieved, as compared with lactic acid fermentation using electrodialysis wastewater supplemented with glucose only.

Lactic acid production from agricultural resources as cheap raw materials.

Agricultural resources such as barley, wheat, and corn were hydrolyzed by commercial amylolytic enzymes and fermented into lactic acid by Enterococcus faecalis RKY1. Although no additional nutrients were supplemented to those resources, lactic acid productivities were obtained at >0.8 g/l h from barley and wheat. When 200 g/l of whole wheat flour was hydrolyzed by amylolytic enzymes after the pre-treatment with 0.3% (v/v) sulfuric acid and sterilized by filtration, E. faecalis RKY1 efficiently produced lactic acid with 2.6 g/l h of lactic acid productivity and 5.90 g/l of maximal dry cell weight without additional nutrients. Lactic acid productivity and cell growth could be enhanced to 31% and 12% higher values than those of non-adapted RKY1, by adaptation of E. faecalis RKY1 to CSL-based medium. When the medium contained 200 g/l of whole wheat flour hydrolyzate, 15 g/l of corn steep liquor, and 1.5 g/l of yeast extract, lactic acid productivity and maximal dry cell weight were obtained at 5.36 g/l h and 14.08 g/l, respectively. This result represented an improvement of up to 106% of lactic acid productivity and 138% of maximal dry cell weight in comparison to the fermentation from whole wheat flour hydrolyzate only.

Culture medium optimization for acetic acid production by a persimmon vinegar-derived bacterium.

A new acetic acid-producing microorganism, Acetobacter sp. RKY4, was isolated from Korean traditional persimmon vinegar, and we optimized the culture medium for acetic acid production from ethanol using the newly isolated Acetobacter sp. RKY4. The optimized culture medium for acetic acid production using this microorganism was found to be 40 g/L ethanol, 10 g/L glycerol, 10 g/L corn steep liquor, 0.5 g/L MgSO4.7H2O, and 1.0 g/L (NH4)H2PO4. Acetobacter sp. RKY4 produced 47.1 g/L of acetic acid after 48 h of fermentation in a 250 mL Erlenmeyer flask containing 50 mL of the optimized medium.

Production of antioxidant compounds by culture of Panax ginseng C.A. Meyer hairy roots: I. Enhanced production of secondary metabolite in hairy root cultures by elicitation.

Ginseng (Panax ginseng C.A. Meyer) hairy root cultures, established by infecting ginseng root discs with Agrobacterium rhizogenes, were used for secondary metabolite production. In this study, several elicitors [salicylic acid (SA), acetylsalicylic acid (ASA), yeast elicitor, and bacterial elicitor] were used to improve the productivity of useful metabolite in P. ginseng hairy root cultures. In SA elicitation, total ginseng saponin content increased slightly at lower elicitor dosages (0.1 to 0.5 mM). Also, the use of ASA as an elicitor resulted in the inhibition of biomass growth and an increase in total ginseng saponin content at every elicitor dosage (0.1 to 1.0 mM) by about 1.1 times. With yeast elicitor addition, hairy root growth was inhibited about 0.8-fold on a dry weight basis compared to the control, but total ginseng saponin content increased by about 1.17 times when compared to the control. The bacterial elicitor showed a slight inhibition of biomass growth, but total ginseng saponin content increased by about 1.23 times upon the addition of 1 mL.

Fermentative production of DL-lactic acid from amylase-treated rice and wheat brans hydrolyzate by a novel lactic acid bacterium, Lactobacillus sp.

Rice and wheat brans, without additional nutrients and hydrolyzed by alpha-amylase and amyloglucosidase, were fermented to DL-lactic acid using a newly isolated strain of Lactobacillus sp. RKY2. In batch fermentations at 36 degrees C and pH 6, the amount of lactic acid in fermentation broth reached 129 g l(-1) by supplementation of rice bran with whole rice flour. The maximum productivity was 3.1 g lactic acid l(-1) h(-1) in rice bran medium supplemented with whole rice flour or whole wheat flour.

Effects of inoculum conditions on growth of hairy roots of Panax ginseng C.A. Meyer.

Plants have a potential to produce a large number of important metabolites such as pharmaceuticals, food additives, pigments, flavors, fragrances, and fine chemicals. Large-scale plant cell and tissue cultures for producing useful products has been considered an attractive alternative to whole plant extraction for obtaining valuable chemicals. In plant cell and tissue cultures, cell growth and metabolite production are influenced by nutritional and environmental conditions as well as physical properties of the culture system. To obtain a high growth rate of plant cell and tissue cultures, the culture conditions should be maintained at an optimum level. We studied the relationship between inoculum conditions and the growth of Panax ginseng hairy root culture, and found that the growth rate varied with the inoculum conditions such as the number of root tips, the length of root tips, the part of root tips, and the inoculum size and age of hairy roots.

Production of fumaric acid using rice bran and subsequent conversion to succinic acid through a two-step process.

The fungal production of fumaric acid using rice bran and subsequent bacterial conversion of succinic acid using fungal culture broth were investigated. Since the rice bran contains abundant proteins, amino acids, vitamins, and minerals, it is suitable material that fungi use as a nitrogen source. The effective concentration of rice bran to produce fumaric acid was 5 g/L. A large amount of rice bran caused excessive fungal growth rather than enhance fumaric acid production. In addition, we could produce fumaric acid without the addition of zinc and iron. Fungal culture broth containing approx 25 g/L of fumaric acid was directly employed for succinic acid conversion. The amount of glycerol and yeast extract required for succinic acid conversion was reduced to 70 and 30%, respectively, compared with the amounts cited in previous studies.

Biotechnological production of L(+)-lactic acid from wood hydrolyzate by batch fermentation of Enterococcus faecalis.

The inhibition of lactic acid fermentation by wood hydrolyzate was decreased (approx. 20%) by adaptation of Enterococcus faecalis RKY1 to wood hydrolyzate-based medium whereby lactic acid productivity and cell growth were enhanced by 0.5 g l(-1) h(-1) and 2.1 g l(-1), respectively. When the diluted or concentrated wood hydrolyzate (equivalent to 25-100 g glucose l(-1)) was supplemented with 15 g yeast extract l(-1), 24-93 g lactic acid l(-1) was produced at a rate between 1.7 g l(-1) h(-1) and 3.2 g l(-1) h(-1).